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Abstract:

A cleaning medium or formulation that contains a sporicidal composition
is described. The composition includes about 0.1-20% weight/weight of a
germinant agent, about 0.01-75% w/w of an antimicrobial agent, in terms
of dry or wet total weight, and which is admixed with water to generate a
solution with a pH of 3.5-9.5. The composition can help trigger the
germination of spores, in particular C. difficile, and subsequently
deactivate or kill the spores. A means of applying the cleaning
formulation in a medium and process for cleaning are also described.

Claims:

1. A cleaning medium or formulation containing a sporicidal composition
comprising: about 0.1-20% weight/weight of a germinant agent, about
0.01-75% w/w of an antimicrobial agent, in terms of dry or wet total
weight, and which is admixed with water to generate a solution with a pH
of 3.5-9.5.

2. The cleaning medium according to claim 1, wherein said germinant agent
is present in an amount from about 1.0% w/w to about 18% w/w, and said
antimicrobial agent is present in an amount from about 0.1% w/w to about
62% w/w.

3. The cleaning medium according to claim 1, wherein said germinant agent
is one of the following: sodium taurocholate, glycocholate, cholate,
glycine, or a combination thereof.

10. The cleaning medium according to claim 5, wherein said surfactant is
at least one of the following: anionic, cationic, non-ionic, and
amphoteric, or a combination thereof.

11. The cleaning medium according to claim 1, wherein said sporicidal
composition exhibits at least a 90% reduction of live Clostridium
difficile spores within about 10 minutes of application of said cleaning
medium to a spore-contaminated surface.

12. The cleaning medium according to claim 10, wherein said sporicidal
composition exhibits at least a 90% reduction of live C. difficile spores
within about 1 minute of application to a spore-contaminated surface.

13. The cleaning medium according to claim 1, wherein said sporicidal
composition further includes up to about 25% w/w of a reducing agent, or
up to about 2% w/w of an electron transport accelerator.

15. The cleaning medium according to claim 13, wherein said electron
transport accelerator is one of the following: phenazine methosulfate,
phenazine ethosulfate, 7-hydroxycoumarin, vanillin,
p-hydroxybenzenesulfonate, and methylene blue.

16. A wiper comprising a substrate sheet; a sporicidal composition
disposed over or within at least part of said sheet, said sporicidal
composition containing about 0.1-20 w/w % of a germinant agent, about
0.01-75% w/w of a antimicrobial agent, in terms of dry total weight,
optionally up to about 10% w/w of a protein denaturant, and up to about
10 w/w % of a surfactant, with water to generate a solution with pH of
3.5-9.5.

17. The wiper according to claim 16, wherein said sporicidal composition
further includes up to about 25% w/w of a reducing agent, or up to about
2% w/w of an electron transport accelerator.

18. The wiper according to claim 16, wherein said substrate sheet is
formed from either a cellulose-based material or nonwoven web.

19. The wiper according to claim 16, wherein said substrate sheet is
formed with a material selected from at least one of the following: a
cellulose-based fibrous tissue, a meltblown, hydroknit, coform, or
spunlace nonwoven, or a combination of cellulose and synthetic polymer
fibers.

20. The wiper according to claim 16, wherein said substrate exhibits a
spore population kill rate of at least 1 Log10 within about 10
minutes of when said wiper is applied to a spore-contaminated surface.

21. A cleaning process for killing Clostridium difficile spores, the
process comprising: providing a cleaning medium with a sporicidal
composition, said sporicidal composition containing about 0.1-20% w/w of
a germinant agent, about 0.01-70% w/w of a antimicrobial agent, in terms
of dry total weight, and which is admixed with water to generate a
solution with a pH of 3.5-8.5; heating said sporicidal composition up to
about 45.degree. C.; and applying said cleaning medium to a
spore-contaminated surface.

22. The cleaning process according to claim 21, further comprising mixing
said heated sporicidal composition.

23. The cleaning process according to claim 21, wherein said mixing is by
means of applying ultrasonic energy of up to 500 kHz to said cleaning
medium.

24. The cleaning process according to claim 21, wherein said
spore-contaminated surface is part of a piece of furniture, table or
countertop, floor, wall, bath or lavatory surfaces, bedclothes, and
linens.

25. The cleaning process according to claim 21, wherein said
spore-contaminated surface is human skin

26. The cleaning process according to claim 21, wherein said
spore-contaminated surface is part of a medical device or instrument.

27. The cleaning process according to claim 21, wherein said application
of said cleaning medium is by immersion bath, wiping or washing.

Description:

FIELD OF INVENTION

[0001] The present invention relates to a cleaning medium that includes a
sporicidal chemical formulation. In particular, the invention pertains to
a composition that can help trigger the germination of spores, and
subsequently deactivate or kill the spore. A means of applying the
cleaning formulation in a medium is also described.

BACKGROUND

[0002] Spore forming Clostridium difficile associated diseases (CDAD)
remain an important nosocomial infection associated with significant
morbidity and mortality. In recent years, the incidence of infection by
this condition has unfortunately increased and high rates of recurrent
disease continue with currently available treatment regimens. Typically,
Clostridium difficile is transmitted by the fecal-oral route. Spores that
persist in the environment survive the gastric acid barrier and germinate
in the colon. Toxins released from vegetative C. difficile cells are
responsible for clinical CDAD.

[0003] As a spore former, C. difficile is more difficult to eradicate than
other bacteria because of its dormant spore state. Although vegetative C.
difficile can only survive 15 minutes aerobically, they are resilient
because they form spores. C. difficile spores can be found as airborne
particles, attached to inanimate surfaces such as hard surfaces and
fabrics, and animate surfaces such as skin and hair. Spores can be found
on the patient's skin as well as any surface in the room that the
infected patient occupied. During exams these spores can be transferred
to the hands and body of healthcare workers and therefore spread to all
subsequent equipment and areas they contact.

[0004] Hospital discharges for CDAD in the United States doubled between
1996 and 2003. These nosocomial infections are extremely costly to
hospitals at $1.28 to $9.55 billion annually in the U.S. alone, mostly
due to infected patients requiring extended stays of 3.6 to 14.4 days.
Complications of CDAD include life-threatening diarrhea,
pseudo-membranous colitis, toxic megacolon, sepsis, and death. Expenses
related to treatment of these conditions ranges from $3,669 to $27,290
per patient. CDAD causes death in 1-2% of affected patients.

[0005] People are most often infected in hospitals, nursing homes, or
institutions, although C. difficile infection in the community,
outpatient setting is increasing. C. difficile infection (CDI) can range
in severity from asymptomatic to severe and life-threatening, especially
among the elderly. The rate of C. difficile acquisition is estimated to
be 13% in patients with hospital stays of up to 2 weeks, and 50% in those
with hospital stays longer than 4 weeks.

[0006] While currently available antibiotics used for treatment of
recurrent CDAD lead to symptomatic improvement, they are ineffective
against C. difficile spores, the transmissible form of the disease. This
causes a high risk of relapse occurring post-therapy as sporulated
microorganisms begin to germinate. Therefore, controlling C. difficile
infection requires limiting the spread of spores by good hygiene
practices, isolation and barrier precautions, and environmental cleaning.

[0007] Because of the prevalence of C. difficile in hospitals, healthcare
workers and researchers have an interest in developing a sporicidal agent
that can kill C. difficile and its spores. Currently, harsh chemicals
have been used, such as bleach, alkyating agents, and acids to kill
spores on surfaces. These commercial technologies are not appealing due
to their high carcinogenic and corrosive nature. Therefore, a need exists
to develop an alternative chemical composition that is as effective as
current sporicidal agents but is gentle on the skin and to the
environment. Mixing a skin safe biocide with a germinant may meet this
need.

[0008] Bile salts have been reported to significantly increase spores
recovery from environmental surfaces and stool. Recent in vitro studies
showed that sodium taurocholate and glycine were cogerminants for C.
difficile spore germination. A new sporicidal formulation containing
germinants that can be incorporated into a cleaning solution can greatly
enhance the control of C. difficile spores. The cleaning solution,
associated supplies, and/or cleaning techniques can benefit sanitation
workers in their efforts to maintain a germ-free environment when
cleaning possible contaminated surfaces.

SUMMARY OF THE INVENTION

[0009] The present invention, in part, pertains to a cleaning formulation,
solution, or dry powder that contains a sporicidal composition
comprising: about 0.1-20% weight/weight (% w/w) of a spore germinant
agent, about 0.01-70% or 75% w/w of an antimicrobial agent, in terms of
wet or dry total weight, and which can be mixed with water to generate a
solution with a pH of 3.5-8.5 or 9.5. This synergistic composition
appears to trigger germination, either concurrently or sequentially with
spore inactivation. When bacterial spores are exposed to a suitable
germinant which triggers the initiation of germination, they are
significantly more susceptible to antimicrobials.

DETAILED DESCRIPTION OF THE INVENTION

[0010] Clostridium difficile, also known as "CDF/cdf", or "C. diff", a
species of Gram-Positive, spore-forming anaerobic bacillus, can lead to
severe complications ranging from antibiotic-associated diarrhea (AAD) to
severe life-threatening pseudomembranous colitis, a severe infection of
the colon. In fact, C. difficile is the cause of approximately 25% of all
cases of antibiotic-associated diarrhea. Most cases of C. difficile
associated disease (CDAD) occur in hospitals or long-term care facilities
causing more than 300,000 cases per year in the United States alone. The
total US hospital costs for CDAD management have been estimated to be
$3.2 billion per year.

[0011] Clostridia are motile bacteria that are ubiquitous in nature and
are especially prevalent in soil. Under microscopy, clostridia appear as
long drumstick-like irregularly-shaped cells with a bulge at their
terminal ends. Clostridium difficile cells show optimum growth on blood
agar at human body temperatures in the absence of oxygen. When stressed,
the bacteria produce spores, which tolerate extreme conditions that the
active bacteria cannot tolerate.

[0012] In small numbers, C. difficile do not result in significant
disease. The first step in development of C. difficile colonization is
the disruption of the normal flora of the colon, usually by antibiotics.
Antibotic treatments, especially those with a broad spectrum of activity,
cause disruption, often resulting from eradication of the normal
intestinal flora by antibiotics of normal intestinal flora, leading to an
overgrowth of C. difficile. C. difficile is currently the most common
cause of nosocomial diarrhea with significant morbidity and mortality.
The C. difficile bacteria, which naturally reside in the human
intestines, overpopulate and release toxins that can cause bloating,
constipation, or diarrhea with abdominal pain, which may become severe.
Latent symptoms often mimic some flu-like symptoms.

[0013] Antibiotic treatment of C. difficile infections can be difficult,
due both to antibiotic resistance as well as physiological factors of the
bacteria itself. Because the organism forms acid and heat-resistant
spores, C. difficile spores can persist in the environment for years and
contamination by C. difficile is very common in hospital, clinical,
long-term care or nursing home environments. Often, it can be cultured
from almost any surface in a hospital. Patient-to-patient transmission of
C. difficile spores occurs by sharing the medical equipments or
facilities in hospitals, nursing homes, and other extended-care
facilities. Typically, C. difficile is transmitted from person to person
by the fecal-oral route. Ingested spores of C. difficile survive the
gastric acid barrier and germinate in the colon. Vegetative cells release
two potent toxins that ultimately mediate diarrhea and colitis.

[0014] Given the pathogenesis of C. difficile, judicious use of
antibiotics and strict infection control and environmental measures are
keys to the prevention of disease. The implementation of antibiotic
stewardship programs has been associated with decreased incidence of
CDAD. To prevent spread of spores, environmental cleaning and patient
isolation are needed. Several disinfectants commonly used in hospitals
may be ineffective against C. difficile spores, and may actually promote
spore formation. However, disinfectants containing bleach are effective
in killing the organisms.

[0015] Health care workers should avoid of using alcohol hand rubs,
especially in outbreak settings, because alcohol is not effective at
killing clostridia spores. Due to their resistant nature, spores are very
difficult to be eliminated with standard measures. Consumer and health
care applications are taking extreme approaches with harsh chemicals
including aldehydes and highly reactive oxidizing agents which are either
carcinogenic or corrosive. It would be virtually impossible to use
current technologies on skin and delicate devices. There is a need to
develop a disinfectant that is nonreactive to untargeted materials and
nonharmful to humans and environment.

Section I--Sporicidal Composition

[0016] The present invention, in part, describes a sporicidal composition
that is effective against C. difficile spores. The composition contains
at least two major components, desirably at least three: a) a C.
difficile specific germinant which binds to the germination receptor to
initiate spore germination; b) a surfactant which may facilitate
transport of biocide and/or germinant across the membrane; and c) a
biocide which inactivates the spore by multiple mechanisms, such as
either disrupting membranes or inactivating essential cellular functions.
It is believed that as soon as germination is triggered, water influx and
Ca+2-dipicolinate release from the spore core takes place. An
increased exchange of flow in and out of spores coat may facilitate
transport of surfactant and biocide through spore cortex. These three
components may work together to deliver synergistic sporicidal effects.

[0017] The sporicidal composition includes, on a dry or wet weight basis,
about 0.1-17% or 20% weight/weight of a germinant agent, about 0.01-65%
w/w, or 70% or 75% w/w of an antimicrobial agent, and which can be mixed
with water to generate a solution with a pH of 3.5-9.5, desirably about
pH 4 or 5-8.0, 8.5 or 9.0. The germinant agent can be present in an
amount from about 1.0, 2.0%, or 3.5% w/w to about 15%, 18%, or 20% w/w,
and the antimicrobial agent is present in an amount from about 0.1%,
0.5%, or 1% w/w to about 60% or 62% w/w. The composition may further
include up to about 8%, 10% or 12% w/w of a protein denaturant, up to
about 8%, 10%, or 12% w/w of a surfactant, up to about 23% or 25% w/w of
a reducing agent, and/or up to about 1.5% or 2% w/w of an electron
transport accelerator. The protein denaturant can be present in an amount
from about 0.1%, 0.5%, or 1% w/w to about 7%,8%, 9.0% or 10% w/w. The
surfactant can be present in an amount from about 0.5%, 0.7% or 1% w/w to
about 7% or 8.7% w/w. When dissolved in an aqueous or polar organic
solvent, the active concentrations of the active ingredients may range
from 0.1-95% w/w, including all ingredients. Typically, the range may be
from about 0.5%, 1%, 1.5% or 2.5% w/w to about 70%, 75%, 80% or 85% w/w,
inclusive of all permutations and combinations thereinbetween.

[0018] The germinant agent, for example, can be one of the following:
sodium taurocholate, glycocholate, cholate, glycine, or a combination
thereof. The antimicrobial agent, for instance, can be one of the
following: an alcohol, quaternary ammonium compounds, biguanides,
triclosan, peroxides, hypochlorites, hypochlorous acid, iodine, silver,
copper, isothiazalones, short-chain acids, or a combination thereof. The
protein denaturant, for example, can be one of the following: urea,
sodium lauryl sulfate, guanidine hydrochloride, ethylene-diamine
tetra-acetic acid, acetic acid, alcohol, aldehydes,
tris(2-carboxyethyl)phosphine, or a combination thereof. The surfactant,
for instance, can be one of the following: anionic, cationic, non-ionic,
and amphoteric, or a combination thereof. The reducing agent, for
instance, can be one of the following: sodium thioglycollate, cysteine,
zinc, copper, nickel, magnesium, manganese, ferrous iron, sulfite
compounds, di-isobutylaluminum hydride, alcohols, sugar alcohols,
titanium, amorphous ferrous sulfide, sodium borohydride, lycopene, and
vitamin E. The electron transport accelerator, for example, can be one of
the following: phenazine methosulfate, phenazine ethosulfate,
7-hydroxycoumarin, vanillin, p-hydroxybenzenesulfonate, and methylene
blue.

[0019] The sporicidal composition exhibits at least a 90% reduction of
live Clostridium difficile spores within about 10-15 minutes of
application of said cleaning medium to a spore-contaminated surface. In
desirable embodiments, the sporicidal composition can be at least 90%
efficient at reducing live C. difficile spores within about 1 minute of
application to a spore-contaminated surface. If the antimicrobial agent
is an alcohol, its concentration should be >62% w/w of dry or wet
total weight.

Section II--Cleaning Substrate

[0020] In another aspect, the present invention relates to a wiper or
sheet. The wiper has a substrate sheet; a sporicidal composition disposed
over or within at least part of said sheet, said sporicidal composition
containing about 0.1-18% or 20% w/w of a germinant agent, about 0.01-70%
or 75% w/w of a antimicrobial agent, in terms of dry or wet total weight,
optionally up to about 10% w/w of a protein denaturant, up to about 10%
w/w of a surfactant, up to about 25% w/w a reducing agent, and/or up to
about 2% w/w of an electron transport accelerator with water to generate
a solution with a pH of 3.5-8.5. Other ingredients, such as reducing
agent or electron transporter, may also be included.

[0021] Furthermore, it is possible to likewise incorporate a dry
formulation into or on a wipe, to deliver sporicidal actives to skin or
other surface that has been pre-wetted. The wiper substrate sheet can be
formed from either a cellulose-based material or nonwoven web. In
particular, the substrate sheet can be formed with a material selected
from at least one of the following: a cellulose-based fibrous tissue, a
meltblown, hydroknit, coform, or spunlace nonwoven, or a combination of
cellulose and synthetic polymer fibers. The wiper substrate also can
exhibit a spore population kill rate of at least 90% or 1 Log10,
within about 15 minutes (typically within about 10 minutes, or desirably
under about 5-7 minutes) of when said wiper is applied to a
spore-contaminated surface.

[0022] Alternatively, a dry wipe impregnated with the composition
described herein could be utilized as well. Water can then be added to
wipe upon use of the product to activate the cleaning formulation. For
example, upon dispensing from a package, one could wet or immersed a wipe
sheet in water and then used it to clean the desired surface.
Alternatively, the surface desired to be cleaned can be sprayed or
pre-treated with water prior to cleaning with the wipe.

[0023] Wiper embodiments may have substrate materials that are selected
from either woven or nonwoven fabrics. Woven fabrics may be made from
natural fibers (e.g., cellulose, cotton, flax linen, hemp, jute, wool,
silk) or a blend of natural and synthetic fibers (e.g., thermoplastics,
polyolefin, polyester, nylon, aramide, polyacrylic materials). A wide
variety of elastic or non-elastic thermoplastic polymers may be used to
construct nonwoven substrate materials. For example, without limitation,
polyamides, polyesters, polypropylene, polyethylene, copolymers of
ethylene and propylene, polylactic acid and polyglycolic acid polymers
and copolymers thereof, polybutylene, styrenic co-block polymers,
metallocene-catalyzed polyolefins, preferably with a density of less than
0.9 gram/cm3, and other kinds of polyolefins, for the production of
various types of elastic or non-elastic fibers, filaments, films or
sheets, or combinations and laminates thereof.

[0024] One may use a wipe sheet to clean various different kinds of
surfaces either in a clinical or other type of setting. These may
include, for instance, various desk, table or countertops or other parts
of furniture surfaces, bath and lavatory surfaces, floor and wall
surfaces, medical instruments or devices, or even human skin or bedding
and linens. In a liquid form, the present composition may be employed in
bath or rinse to wash medical instruments, linens, bedclothes, or human
skin. One may even incorporate use the formulation in a disinfecting or
sanitary solution to wash hands or medical instruments.

[0025] According to the invention, we envision that one could use heat
and/or sonication as part of the spore inactivation process. A heated wet
wipe or a sponge may be employed in conjunction with an ultrasonic device
during the cleaning process. The heating element and/or sonication device
either may be integrated as part of the wipe or sponge cleaning tools, or
can be separate stand-alone or secondary devices. Furthermore, an
ultrasonic device can be used to enhance the inactivation process alone,
without heat. Each of the two processes may be beneficial independently
or combined. In an embodiment, a heater may be activated to raise the
temperature of the wipers in a wipes container before use, or one may
incorporate exothermic ingredients that warm the wipe upon use. For
instance, some ingredients may be exothermically activated when
interacting by friction against the surface to be cleaned or by microwave
irradiation. Some exothermic materials may include, for example, oxidized
iron powder, electrolyte salts (e.g., magnesium chloride), electrical
sources, infrared, and microwave radiation. Some heating agents may be
microencapsulated to increase their stability during processing and prior
to use.

[0026] In certain embodiments, the sporicidal compositions can be applied
topically to the external surfaces of nonwoven web filaments after they
are formed. Desirably, a uniform coating is applied over the filament
substrate surfaces. A uniform coating refers to a layer of the
formulation that does not aggregate only at selected sites on a substrate
surface, but has a relatively homogeneous or even distribution over the
treated substrate surface. Desirably, the processing aid should evaporate
or flash off once the cleaning composition dries on the substrate
surface. Suitable processing aids may include alcohols, such as hexanol
or octanol. Note that the terms "surface treatment," "surface
modification," and "topical treatment" refer to an application of the
present formulations to a substrate and are used interchangeably, unless
otherwise indicated.

[0027] Nonwoven fabrics that are treated with a coating of the present
invention can be fabricated according to a number of processes. According
to an embodiment, the present composition can be applied to the material
substrate via conventional saturation processes such as a so-called "dip
and squeeze" or "padding" technique. The "dip and squeeze" or "padding"
process can coat both sides of and/or through the bulk of the substrate
with the sporicidal composition. When dipped in a bath, the formulation
can be a unitary medium containing all components, or in subsequent
multiple step processing, other desired components may be later added to
the base layer. On substrates containing polypropylene, an antistatic
agent can help dissipate static charge build-up from mechanical friction.
An antistatic agent can be added to the sporicidal solution, and the
mixture can be introduced simultaneously to the material substrate in one
application step. Alternatively, the antistatic solution can be applied
using a spray after the sporicidal formulation in a second step. In
certain product forms, where one wishes to treat only a single side and
not the inner layers or opposing side of the sheet substrate, in which
the substrate material is layered to another sheet ply (e.g., filter or
barrier media) that is without the sporicidal treatment, other processes
are preferred such as at rotary screen, reverse roll, Meyer-rod (or wire
wound rod), Gravure, slot die, gap-coating, or other similar techniques,
familiar to persons in the nonwoven textile industry. (See, for example,
detailed descriptions of these and other techniques are available from
Faustel Inc., Germantown, Wis. (www.faustel.com).) Also one may consider
printing techniques such as flexographic or digital techniques.
Alternatively one may use a combination of more than one coating to
achieve a controlled placement of the treatment composition. Such
combination may include, but not limited to, a reverse Gravure process
followed by a Meyer rod process. Alternatively, the composition may be
applied through an aerosol spray on the substrate surface. The spray
apparatus can be employed to apply the antimicrobial cleaning solution
and/or antistatic agent only on one side of the substrate sheet or on
both sides separately if desired.

Section III--EXAMPLES

[0028] The following describes a protocol for determining the killing
efficacy of the present cleaning formulation or medium.

Procedure:

[0029] 1. Prepare a spore suspension of C. difficile to 105 to
107 CFU/ml in Phosphate Buffered Saline (PBS). [0030] 2. Add 100
μl of spore culture to sterile vial containing 900 μl of test
solution and vortex. [0031] 3. At the desired time point, vortex the
vials. [0032] 4. Add 100 μl of test solution and spore mixture to
sterile tubes containing 900 μl of neutralizer. [0033] 5. Place the
neutralized samples in a sonicating water bath for five cycles of 1
minute on, 1 minute off. [0034] 6. Vortex each tube and pipette 100 μl
of the solution on BHI+0.15% sodium taurocholate media plates (prepared
in lab according to dehydrated powder instructions). [0035] 7. Prepare a
control sample by adding the spore culture to 900 μl of PBS and repeat
steps 3-6 above. [0036] 8. Place plates in anaerobic jars or boxes or in
an anaerobic chamber and incubate for 48±4 hours at 37±3° C.
[0037] 9. After incubation, enumerate colonies and record results.
Calculate Log10 reduction by comparing the number of colonies
recovered from the test solution versus those recovered with the control.
The results summarized in the following Examples demonstrated that
antimicrobial agents alone or in combination with a surfactant had little
effect on inactivating C. difficile spores. However, when in combination
with a germinant(s), one observed synergistic sporicidal efficacy.
Additionally, a similar synergistic effect may be possible via the
combination of a germinant(s), surfactant(s), reducing agent(s), electron
transport accelerator(s), and protein denaturant(s).

[0038] The present invention has been described in general and in detail
by way of examples. Persons of skill in the art understand that the
invention is not limited necessarily to the embodiments specifically
disclosed, but that modifications and variations may be made without
departing from the scope of the invention as defined by the following
claims or their equivalents, including other equivalent components
presently known, or to be developed, which may be used within the scope
of the present invention. Therefore, unless changes otherwise depart from
the scope of the invention, the changes should be construed as being
included herein.